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Cited by 398Mitochondrial Function and PathologyMuscle Physiology and DisordersAutophagy in Disease and Therapy

The DOI matches its Crossref record; 62 of the 62 references it cites were found in a registry or an index; 4 of the 27 sources its claims cite were checked for support.

DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass

Open access
Favaro, Giulia, Romanello, Vanina, Varanita, Tatiana, Andrea Desbats, Maria, Morbidoni, Valeria, Tezze, Caterina, Albiero, Mattia, Canato, Marta, Gherardi, Gaia, De Stefani, Diego, Mammucari, Cristina, Blaauw, Bert, Boncompagni, Simona, Protasi, Feliciano, Reggiani, Carlo, Scorrano, Luca, Salviati, Leonardo, Sandri, Marco·2019·Nature Communications
DOI 10.1038/s41467-019-10226-9PMC PMC6561930
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Abstract

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca 2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca 2+ handling.

Overview

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TL;DR

Muscle-specific loss of DRP1 in mice causes a lethal mitochondrial myopathy with atrophy, weakness, and altered calcium homeostasis. DRP1-deficient mitochondria are larger and dysfunctional, triggering UPR/UPS activation, blocked autophagy/mitophagy, and increased MCU-mediated Ca²⁺ uptake that contributes to myofiber death.

Key findings

  • Constitutive muscle-specific Drp1 deletion caused postnatal lethality within 30 days (WT n = 43; KO n = 14), reduced body growth, fewer glycolytic 2B/2X fibers, smaller cross-sectional area, and a 30% reduction in protein synthesis.
  • Inducible adult Drp1 deletion reduced fiber size by 25% at 70 days and 50% at 180 days, decreased absolute and specific force, and produced center-nucleated fibers in 10% of tibialis anterior and 25% of gastrocnemius myofibers at 180 days.
  • Drp1-deficient mitochondria were larger and fewer with unchanged mtDNA content and mitochondrial mass, but showed reduced respiration (complex-I-driven respiration most affected), decreased respiratory chain supercomplexes, increased OPA1 cleavage, and in acute deletion about 10% damaged mitochondria versus 2% in WT.
  • DRP1 loss activated UPR (Bip/Grp78, p-eIF2α, ATF4, FGF21; FGF21 blood increased 20-fold in acute KO), induced FoxO3/UPS, blocked autophagy/mitophagy, and increased mitochondrial Ca²⁺ uptake via MCU upregulation and miR1 downregulation; MCU knockdown normalized cytosolic Ca²⁺ and reduced center-nucleated fibers.

Methods

  • Two muscle-specific Drp1 knockout models: constitutive Drp1fl/fl × MLC1f-Cre mice analyzed at postnatal day 12, and tamoxifen-inducible Drp1fl/fl × HSA-Cre-ER mice treated at 5 months of age; Cre-negative littermates served as controls.
  • Readouts included electron microscopy/stereology, histology and SDH staining, RT-qPCR, immunoblotting, mitochondrial respiration and Blue Native PAGE, SUnSET protein synthesis, TMRM membrane potential, Fura-2 and cameleon Ca²⁺ probes, in vivo/ex vivo force and fatigue measurements, mito-Keima mitophagy assay, MCU knockdown, and plasma FGF21/IGF1 measurements.
  • Data are means ± SEM; statistical analysis used two-tailed unpaired Student’s t test or two-way ANOVA with Bonferroni’s multiple comparison test, with significance at p < 0.05.

Limitations

  • Stated: MCU knockdown did not completely block degeneration/regeneration because transfection efficiency was low, so MCU was reduced only in some myofibers.
  • Evident: all findings are from mouse models; no human patient validation is reported.
  • Evident: some analyses used very small n (e.g., WT n = 2 for succinate/rotenone respiration in Fig. 5d), and the causal role of Ca²⁺ dysregulation is supported by rescue/knockdown experiments but not fully isolated from other DRP1-dependent defects.

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Registry

Crossref · 10.1038/s41467-019-10226-9

Cites

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Figures

Fig. 1 DRP1 deletion impairs muscle growth and causes a lethal phenotype. Drp1 mRNA (a) and DRP1 protein levels (b) are downregulated only in skeletal muscle and not in other tissues in DRP1-null mice. Each condition represents the average of at least three independent experiments ± SEM. c Kaplan–Meier survival curve of Drp1fl/fl and Drp1−/− littermates (WT, n = 43; KO, n = 14), indicates that muscle-specific DRP1 deletion results in lethality within postnatal day 30. d DRP1-deficient mice show an impairment of total body weight. e Upper panel: representative photograph of control and KO littermates at postnatal day 12 showing that Drp1−/− mice are smaller than controls. Lower panel: haematoxilin–eosin (HE) staining of hindlimb cross-section. The cross-sectional area analysis of fibers (g) and the total number of different fiber types in hindlimb muscles (f) indicate a reduction of DRP1 KO fiber size without hypoplasia. The data represent mean ± SEM (g, n = 3 and f, n = 3). h Representative immunoblot analysis of muscle homogenates of four independent experiments. MyoD and Myogenin were normalized to GAPDH expression levels. The data represent average ± SEM. Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 1 DRP1 deletion impairs muscle growth and causes a lethal phenotype. Drp1 mRNA (a) and DRP1 protein levels (b) are downregulated only in skeletal muscle and not in other tissues in DRP1-null mice. Each condition represents the average of at least three independent experiments ± SEM. c Kaplan–Meier survival curve of Drp1fl/fl and Drp1−/− littermates (WT, n = 43; KO, n = 14), indicates that muscle-specific DRP1 deletion results in lethality within postnatal day 30. d DRP1-deficient mice show an impairment of total body weight. e Upper panel: representative photograph of control and KO littermates at postnatal day 12 showing that Drp1−/− mice are smaller than controls. Lower panel: haematoxilin–eosin (HE) staining of hindlimb cross-section. The cross-sectional area analysis of fibers (g) and the total number of different fiber types in hindlimb muscles (f) indicate a reduction of DRP1 KO fiber size without hypoplasia. The data represent mean ± SEM (g, n = 3 and f, n = 3). h Representative immunoblot analysis of muscle homogenates of four independent experiments. MyoD and Myogenin were normalized to GAPDH expression levels. The data represent average ± SEM. Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001CC BY · PubMed Central
Fig. 2 Muscle DRP1-chronic inhibition alters mitochondrial morphology, RCS organization and complex-I- and complex-II-dependent respiration. a Representative EM cross-sectional images of mitochondrial distribution and morphology in gastrocnemius from WT and Drp1−/− mice. In cross-sections, the different morphology of mitochondria in Drp1−/− from WT is often evident: in Drp1−/− fibers mitochondria are more often larger in size (scale bar: 1 μm). b Mitochondrial DNA copy number quantification. mtDNA was amplified by RT-PCR from the total DNA of gastrocnemius muscles of the indicated genotype. The data are normalized to control and represent average ± SEM of five independent experiments. c Representative immunoblots of four independent experiments of muscle homogenates showing no difference of porin/GAPDH ratio. d RT-PCR analysis of mitochondria dynamics transcripts of muscles of WT and Drp1−/−. DRP1 deletion induces PGC1α, OPA1, and Mfn1 transcript levels. The data represent average ± SEM (n = 8) and are normalized for GAPDH and expressed as fold increase of controls. e Succinate dehydrogenase (SDH) staining of Drp1fl/fl and Drp1−/− muscles show a different distribution of mitochondrial network in KO mice. f Respiratory control ratio (RCR) of muscle-isolated mitochondria energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC. The data represent average ± SEM of three independent experiments. g DRP1 deficiency leads to a significant reduction of CIII and CIV assembly in respiratory chain supercomplexes (RCS). Representative Blue Native PAGE analysis showing RCS developed and normalized for individual respiratory chain complexes. Left panel: CI subunit NDUFB8, middle panel: CIII-Core2 protein 2 and right panel: CIV subunit COXI. h Representative immunoblot analysis of OPA1 different isoforms show an increase in OPA1 cleavage in Drp1-null muscles (n = 4). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 2 Muscle DRP1-chronic inhibition alters mitochondrial morphology, RCS organization and complex-I- and complex-II-dependent respiration. a Representative EM cross-sectional images of mitochondrial distribution and morphology in gastrocnemius from WT and Drp1−/− mice. In cross-sections, the different morphology of mitochondria in Drp1−/− from WT is often evident: in Drp1−/− fibers mitochondria are more often larger in size (scale bar: 1 μm). b Mitochondrial DNA copy number quantification. mtDNA was amplified by RT-PCR from the total DNA of gastrocnemius muscles of the indicated genotype. The data are normalized to control and represent average ± SEM of five independent experiments. c Representative immunoblots of four independent experiments of muscle homogenates showing no difference of porin/GAPDH ratio. d RT-PCR analysis of mitochondria dynamics transcripts of muscles of WT and Drp1−/−. DRP1 deletion induces PGC1α, OPA1, and Mfn1 transcript levels. The data represent average ± SEM (n = 8) and are normalized for GAPDH and expressed as fold increase of controls. e Succinate dehydrogenase (SDH) staining of Drp1fl/fl and Drp1−/− muscles show a different distribution of mitochondrial network in KO mice. f Respiratory control ratio (RCR) of muscle-isolated mitochondria energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC. The data represent average ± SEM of three independent experiments. g DRP1 deficiency leads to a significant reduction of CIII and CIV assembly in respiratory chain supercomplexes (RCS). Representative Blue Native PAGE analysis showing RCS developed and normalized for individual respiratory chain complexes. Left panel: CI subunit NDUFB8, middle panel: CIII-Core2 protein 2 and right panel: CIV subunit COXI. h Representative immunoblot analysis of OPA1 different isoforms show an increase in OPA1 cleavage in Drp1-null muscles (n = 4). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001CC BY · PubMed Central
Fig. 3 DRP1 absence leads to downregulation of protein synthesis and activation of the Ubiquitin–proteasome and autophagy–lysosome pathways. a In vivo SUnSET technique showed a significant reduction of protein synthesis in DRP1-ablated mice muscles. Representative western blot and quantification of the puromycin-labeled peptides, expressed as percentage of the values obtained in the control group, are depicted. The data represent average ± SEM of eight independent experiments. b The total protein extracts from newborns muscles were immunoblotted with the indicated antibodies. A representative immunoblot of four independent experiments is shown. Statistical significance of specified densitometric ratios is indicated on the right. The data are normalized to GAPDH (n = 8). Representative immunoblot (c) and RT-PCR (d) showing the activation of the unfolding protein response (UPR) pathway in DRP1 KO muscles. The data represent average ± SEM of seven independent experiments. e RT-PCR analysis of transcriptional levels of muscle FGF21 (n = 3). f Quantification of blood levels of FGF21 shows an increase in DRP1 KO mice (n = 3), leading to a decrease in blood glucose (g, n = 4) and to a significant decrease in IGF1 class II mRNA level in the liver (h, n = 4). The data represent average ± SEM. i Quantification of IGF1 in plasma from Drp1−/− and control mice shows a significant decrease of IGF1 circulating levels in the absence of DRP1. RT-PCR analysis of FoxO-dependent transcripts shows an increase in FoxO3 levels (j), in the muscle-specific ubiquitin ligases Atrogin1 and MuRF1 (k) and in the novel Ubiquitin–ligase MUSA1 (l). The data represent average ± SEM (n = 6 per condition). m Representative blots and densitometric analysis of at least four independent experiments of total muscle extracts immunoblotted for anti-Ubiquitin (Lys48) and for anti-Ubiquitin (Lys63) normalized to GAPDH. The data represent average ± SEM. n Quantitative PCR analysis of autophagy-related transcripts showing a significant induction of autophagy markers in Drp1−/−. The data represent average ± SEM (n = 8 per condition). o Representative immunoblots of four independent experiments of autophagy-related proteins. The data represent average ± SEM (WT, n = 4; KO, n = 6). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 3 DRP1 absence leads to downregulation of protein synthesis and activation of the Ubiquitin–proteasome and autophagy–lysosome pathways. a In vivo SUnSET technique showed a significant reduction of protein synthesis in DRP1-ablated mice muscles. Representative western blot and quantification of the puromycin-labeled peptides, expressed as percentage of the values obtained in the control group, are depicted. The data represent average ± SEM of eight independent experiments. b The total protein extracts from newborns muscles were immunoblotted with the indicated antibodies. A representative immunoblot of four independent experiments is shown. Statistical significance of specified densitometric ratios is indicated on the right. The data are normalized to GAPDH (n = 8). Representative immunoblot (c) and RT-PCR (d) showing the activation of the unfolding protein response (UPR) pathway in DRP1 KO muscles. The data represent average ± SEM of seven independent experiments. e RT-PCR analysis of transcriptional levels of muscle FGF21 (n = 3). f Quantification of blood levels of FGF21 shows an increase in DRP1 KO mice (n = 3), leading to a decrease in blood glucose (g, n = 4) and to a significant decrease in IGF1 class II mRNA level in the liver (h, n = 4). The data represent average ± SEM. i Quantification of IGF1 in plasma from Drp1−/− and control mice shows a significant decrease of IGF1 circulating levels in the absence of DRP1. RT-PCR analysis of FoxO-dependent transcripts shows an increase in FoxO3 levels (j), in the muscle-specific ubiquitin ligases Atrogin1 and MuRF1 (k) and in the novel Ubiquitin–ligase MUSA1 (l). The data represent average ± SEM (n = 6 per condition). m Representative blots and densitometric analysis of at least four independent experiments of total muscle extracts immunoblotted for anti-Ubiquitin (Lys48) and for anti-Ubiquitin (Lys63) normalized to GAPDH. The data represent average ± SEM. n Quantitative PCR analysis of autophagy-related transcripts showing a significant induction of autophagy markers in Drp1−/−. The data represent average ± SEM (n = 8 per condition). o Representative immunoblots of four independent experiments of autophagy-related proteins. The data represent average ± SEM (WT, n = 4; KO, n = 6). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001CC BY · PubMed Central
Fig. 4 DRP1 loss in adult animals causes body weight loss, muscle atrophy and muscle weakness. a DRP1 protein levels are downregulated in different types of skeletal muscles in HSADRP1-null mice. O.D. levels represent the average of at least three independent experiments ± SEM. b Growth curve of control and Drp1−/− littermates during and after tamoxifen treatment. KO mice start to lose body weight after 7 weeks of tamoxifen treatment and during the following weeks. The data represent average ± SEM (WT, n = 10; KO, n = 11). c Dissected gastrocnemius muscles from the control and DRP1-null mice show an important muscle atrophy after DRP1 deletion in adult animals. d Quantification of the cross-sectional area of myofibers indicates a significant reduction in DRP1-ablated muscles. Values represent average ± SEM (WT, n = 5; KO 70 days, n = 5; KO 180 days, n = 3). e Representative haematoxilin–eosin staining of Tibialis Anterior muscle showing signs of myofiber degeneration/regeneration in Drp1−/− after 180 days of treatment. f In Drp1−/− muscles after 180 days of treatment, there are 10 and 25% of myofibers are centrally nucleated, in tibialis anterior muscle and gastrocnemius muscle, respectively (n = 3 mice each condition). g Force measurements performed in vivo on gastrocnemius muscles. Absence of DRP1 leads to a significant decrease in both absolute force and maximal specific force generated during tetanic contraction. The data represent average ± SEM (n = 3). Two-tailed unpaired Student’s t test and two-way analysis of variant (ANOVA) were used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 4 DRP1 loss in adult animals causes body weight loss, muscle atrophy and muscle weakness. a DRP1 protein levels are downregulated in different types of skeletal muscles in HSADRP1-null mice. O.D. levels represent the average of at least three independent experiments ± SEM. b Growth curve of control and Drp1−/− littermates during and after tamoxifen treatment. KO mice start to lose body weight after 7 weeks of tamoxifen treatment and during the following weeks. The data represent average ± SEM (WT, n = 10; KO, n = 11). c Dissected gastrocnemius muscles from the control and DRP1-null mice show an important muscle atrophy after DRP1 deletion in adult animals. d Quantification of the cross-sectional area of myofibers indicates a significant reduction in DRP1-ablated muscles. Values represent average ± SEM (WT, n = 5; KO 70 days, n = 5; KO 180 days, n = 3). e Representative haematoxilin–eosin staining of Tibialis Anterior muscle showing signs of myofiber degeneration/regeneration in Drp1−/− after 180 days of treatment. f In Drp1−/− muscles after 180 days of treatment, there are 10 and 25% of myofibers are centrally nucleated, in tibialis anterior muscle and gastrocnemius muscle, respectively (n = 3 mice each condition). g Force measurements performed in vivo on gastrocnemius muscles. Absence of DRP1 leads to a significant decrease in both absolute force and maximal specific force generated during tetanic contraction. The data represent average ± SEM (n = 3). Two-tailed unpaired Student’s t test and two-way analysis of variant (ANOVA) were used. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001CC BY · PubMed Central
Fig. 5 Ablation of DRP1 in adulthood modifies mitochondrial shape and reduces RCS assembly, Complex-I-, Complex-II-, and complex-IV-dependent respiration. a SDH staining indicating the presence of bigger mitochondria in KO muscles compared with control. b Representative electron micrographs of EDL muscles of controls and Drp1−/−. Left panel: in WT mitochondria (pointed by black arrowheads) are placed in proximity of Z lines and usually exhibit an electron-dense matrix (inset). Center panel: in Drp1−/− mice mitochondria are often larger in size, oriented longitudinally (large arrows), and damaged (white arrowhead and inset). Right panel: a Drp1−/− fiber presenting spindle-shaped regions (asterisks) which are surrounded by a membrane (inset, white arrow) and that contains vacuoles. c Absence of DRP1 induces mitochondrial depolarization. Isolated adult fibers were loaded with TMRM. Oligomycin and the protonophore FCCP were added at the indicated time points. TMRM staining was monitored in 18 fibers for WT, 52 fibers for KO. Lower panel: representative images of adult myofibers showing altered mitochondrial distribution in DRP1-null muscles. Myofibers were loaded with the potentiometric die TMRM. d Respiratory control ratio (RCR) of muscle isolated mitochondria energized with 5 mM/2.5 mM GLU/MAL or 2 mM rotenone/10 mM succinate or 3 mM ascorbate/10 mM TMPD. The data represent average ± SEM (GLUT/MAL, n = 5 each condition; succinate/rotenone: WT, n = 2; KO, n = 7; ascorbate/TMPD, n = 7 each condition). e The assembly of CIII and CIV in RCS is significantly reduced in mitochondria of DRP1-deficient muscles. Representative Blue Native PAGE analysis showing RCS developed and normalized for individual respiratory chain complexes. CI subunit NDUFB8 (left panel), CIII-Core2 protein 2 (middle panel), and CIV subunit COXI (right panel). f Mitochondrial DNA copy number quantification in controls and KO muscles. mtDNA was amplified by RT-PCR from the total DNA of gastrocnemius muscles of the indicated genotype. The data are normalized to controls and represent the average ± SEM (WT, n = 4; KO, n = 5). g Mitochondrial mass revealed by TOM20 and porin was not affected by DRP1 deletion (n = 4). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; ***p ≤ 0.001
Fig. 5 Ablation of DRP1 in adulthood modifies mitochondrial shape and reduces RCS assembly, Complex-I-, Complex-II-, and complex-IV-dependent respiration. a SDH staining indicating the presence of bigger mitochondria in KO muscles compared with control. b Representative electron micrographs of EDL muscles of controls and Drp1−/−. Left panel: in WT mitochondria (pointed by black arrowheads) are placed in proximity of Z lines and usually exhibit an electron-dense matrix (inset). Center panel: in Drp1−/− mice mitochondria are often larger in size, oriented longitudinally (large arrows), and damaged (white arrowhead and inset). Right panel: a Drp1−/− fiber presenting spindle-shaped regions (asterisks) which are surrounded by a membrane (inset, white arrow) and that contains vacuoles. c Absence of DRP1 induces mitochondrial depolarization. Isolated adult fibers were loaded with TMRM. Oligomycin and the protonophore FCCP were added at the indicated time points. TMRM staining was monitored in 18 fibers for WT, 52 fibers for KO. Lower panel: representative images of adult myofibers showing altered mitochondrial distribution in DRP1-null muscles. Myofibers were loaded with the potentiometric die TMRM. d Respiratory control ratio (RCR) of muscle isolated mitochondria energized with 5 mM/2.5 mM GLU/MAL or 2 mM rotenone/10 mM succinate or 3 mM ascorbate/10 mM TMPD. The data represent average ± SEM (GLUT/MAL, n = 5 each condition; succinate/rotenone: WT, n = 2; KO, n = 7; ascorbate/TMPD, n = 7 each condition). e The assembly of CIII and CIV in RCS is significantly reduced in mitochondria of DRP1-deficient muscles. Representative Blue Native PAGE analysis showing RCS developed and normalized for individual respiratory chain complexes. CI subunit NDUFB8 (left panel), CIII-Core2 protein 2 (middle panel), and CIV subunit COXI (right panel). f Mitochondrial DNA copy number quantification in controls and KO muscles. mtDNA was amplified by RT-PCR from the total DNA of gastrocnemius muscles of the indicated genotype. The data are normalized to controls and represent the average ± SEM (WT, n = 4; KO, n = 5). g Mitochondrial mass revealed by TOM20 and porin was not affected by DRP1 deletion (n = 4). Two-tailed unpaired Student’s t test was used. Statistical significance: *p ≤ 0.05; ***p ≤ 0.001CC BY · PubMed Central
Fig. 6 Protein degradation pathways are activated with acute inhibition of DRP1 in adult muscles. a The total protein extracts from adult muscles were immunoblotted with indicated antibodies. A representative immunoblot of four independent experiments is shown. Statistical significance, of specified densitometric ratios is indicated with asterisks on the right. b In vivo SUnSET technique shows no differences in protein synthesis rate during adulthood (n = 4). c Representative immunoblot (n = 4) and RT-PCR analysis (n ≥ 5) (d) showing the activation of the unfolded protein response and the upregulation of FGF21 in DRP1 KO muscles. e Immunoblot and densitometric quantification showing the accumulation of FoxO3 in the nuclear fraction of tibialis anterior muscles from Drp1−/− mice (n = 5 each condition). f RT-PCR analysis of transcriptional levels of the muscle-specific ubiquitin ligases Atrogin1 and MuRF1 and the novel ubiquitin–ligases MUSA1, SMART, FbxO31, TRIM37, and Itch. The data represent average ± SEM (at least n = 5 per condition). g Quantitative PCR analysis of autophagy-related transcripts showing a significant induction of autophagy markers in Drp1−/−. h Western blot analysis of autophagy marker showing an impaired autophagy in Drp1−/− (WT, n = 4; KO, n = 5). i Immunoblots and relative densitometric quantification from colchicine treated mice support a block in autophagic flux after Drp1 inhibition (n ≥ 5 mice per each condition). j Keima assay indicating the decreased mitophagy in DRP1-KO myofibers (WT, n = 41; KO, n = 39). Two-tailed unpaired Student’s t test was used. Statistical significance: **p ≤ 0.01; ***p ≤ 0.001
Fig. 6 Protein degradation pathways are activated with acute inhibition of DRP1 in adult muscles. a The total protein extracts from adult muscles were immunoblotted with indicated antibodies. A representative immunoblot of four independent experiments is shown. Statistical significance, of specified densitometric ratios is indicated with asterisks on the right. b In vivo SUnSET technique shows no differences in protein synthesis rate during adulthood (n = 4). c Representative immunoblot (n = 4) and RT-PCR analysis (n ≥ 5) (d) showing the activation of the unfolded protein response and the upregulation of FGF21 in DRP1 KO muscles. e Immunoblot and densitometric quantification showing the accumulation of FoxO3 in the nuclear fraction of tibialis anterior muscles from Drp1−/− mice (n = 5 each condition). f RT-PCR analysis of transcriptional levels of the muscle-specific ubiquitin ligases Atrogin1 and MuRF1 and the novel ubiquitin–ligases MUSA1, SMART, FbxO31, TRIM37, and Itch. The data represent average ± SEM (at least n = 5 per condition). g Quantitative PCR analysis of autophagy-related transcripts showing a significant induction of autophagy markers in Drp1−/−. h Western blot analysis of autophagy marker showing an impaired autophagy in Drp1−/− (WT, n = 4; KO, n = 5). i Immunoblots and relative densitometric quantification from colchicine treated mice support a block in autophagic flux after Drp1 inhibition (n ≥ 5 mice per each condition). j Keima assay indicating the decreased mitophagy in DRP1-KO myofibers (WT, n = 41; KO, n = 39). Two-tailed unpaired Student’s t test was used. Statistical significance: **p ≤ 0.01; ***p ≤ 0.001CC BY · PubMed Central
Fig. 7 Acute DRP1 inhibition alters calcium homeostasis. a Cytosolic calcium level at rest is not affected in KO muscles (40 days: WT, n = 24; KO, n = 28; 70 days, n = 19 fibers). b Amplitude of cytosolic Ca2+ transients induced by 0.5 Hz electrical pulses is lower in Drp1−/− mice (40 days, WT, n = 25; KO, n = 28 fibers; 70 days, WT, n = 33; KO, n = 29 fibers). c Cytosolic calcium transients induced by caffeine–thapsigargin are strongly reduced (40 days, WT, n = 10; KO, n = 14 fibers; 70 days, WT, n = 13 fibers; KO, n = 14 fibers). d Mitochondrial matrix-free calcium concentration is not affected at rest but is higher in Drp1−/− compared with control during a train of electrical stimulation at 60 Hz, 2 s duration (WT, n = 9; KO, n = 7 fibers; Peak, n = 6 each condition). e Immunoblot analysis of isolated mitochondria. MCU levels were normalized to TOM20 levels. f Quantitative RT-PCR analysis showing a significant decrease of miR1 levels in Drp1−/− skeletal muscle (n = 4 per condition). The data represent average ± SEM. g Ultrastructure and geometry of CRUs in DRP1 KO muscles. Inserts A and B show T-tubule staining in WT (A) and in DRP1 KO (B); the frequency of oblique/longitudinal tubules increases (stars) when compared with control samples. Inserts C–F are representative EM pictures showing the structural geometry of a normal, an oblique, a longitudinal triad and of a dyad respectively. In inserts A and B, T-tubules are stained in black while in inserts C–F, T-tubules has been false-labeled in green. h Cytosolic calcium transients induced by caffeine–thapsigargin after the reintroduction of DRP1 (Drp1-YFP) in muscle fibers. Cytosolic calcium transients in Drp1−/− in the presence of Drp1-YFP show no difference compared with control fibers (Drp1fl/fl, n = 22; Drp1fl/fl—Drp1-YFP, n = 7; Drp1−/−, n = 22; Drp1−/− – Drp1-YFP, n = 13). i Inhibition of mitochondria Ca2+ uptake via MCU knockdown restored cytosolic Ca2+ levels during caffeine–thapsigargin treatment (WT, n = 17 fibers; WT shMCU, n = 23 fibers; KO, n = 27 fibers; KO shMCU, n = 17 fibers). j MCU inhibition reduces centrally nucleated fibers in KO muscles. For all experiments a minimum of three mice was used for each condition. Two-tailed unpaired Student’s t test and two-way analysis of variant (ANOVA) were used. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 7 Acute DRP1 inhibition alters calcium homeostasis. a Cytosolic calcium level at rest is not affected in KO muscles (40 days: WT, n = 24; KO, n = 28; 70 days, n = 19 fibers). b Amplitude of cytosolic Ca2+ transients induced by 0.5 Hz electrical pulses is lower in Drp1−/− mice (40 days, WT, n = 25; KO, n = 28 fibers; 70 days, WT, n = 33; KO, n = 29 fibers). c Cytosolic calcium transients induced by caffeine–thapsigargin are strongly reduced (40 days, WT, n = 10; KO, n = 14 fibers; 70 days, WT, n = 13 fibers; KO, n = 14 fibers). d Mitochondrial matrix-free calcium concentration is not affected at rest but is higher in Drp1−/− compared with control during a train of electrical stimulation at 60 Hz, 2 s duration (WT, n = 9; KO, n = 7 fibers; Peak, n = 6 each condition). e Immunoblot analysis of isolated mitochondria. MCU levels were normalized to TOM20 levels. f Quantitative RT-PCR analysis showing a significant decrease of miR1 levels in Drp1−/− skeletal muscle (n = 4 per condition). The data represent average ± SEM. g Ultrastructure and geometry of CRUs in DRP1 KO muscles. Inserts A and B show T-tubule staining in WT (A) and in DRP1 KO (B); the frequency of oblique/longitudinal tubules increases (stars) when compared with control samples. Inserts C–F are representative EM pictures showing the structural geometry of a normal, an oblique, a longitudinal triad and of a dyad respectively. In inserts A and B, T-tubules are stained in black while in inserts C–F, T-tubules has been false-labeled in green. h Cytosolic calcium transients induced by caffeine–thapsigargin after the reintroduction of DRP1 (Drp1-YFP) in muscle fibers. Cytosolic calcium transients in Drp1−/− in the presence of Drp1-YFP show no difference compared with control fibers (Drp1fl/fl, n = 22; Drp1fl/fl—Drp1-YFP, n = 7; Drp1−/−, n = 22; Drp1−/− – Drp1-YFP, n = 13). i Inhibition of mitochondria Ca2+ uptake via MCU knockdown restored cytosolic Ca2+ levels during caffeine–thapsigargin treatment (WT, n = 17 fibers; WT shMCU, n = 23 fibers; KO, n = 27 fibers; KO shMCU, n = 17 fibers). j MCU inhibition reduces centrally nucleated fibers in KO muscles. For all experiments a minimum of three mice was used for each condition. Two-tailed unpaired Student’s t test and two-way analysis of variant (ANOVA) were used. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001CC BY · PubMed Central
Fig. 8 Scheme of the mechanisms induced by acute DRP1 inhibition
Fig. 8 Scheme of the mechanisms induced by acute DRP1 inhibitionCC BY · PubMed Central

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Claims the paper makes

36 Own result · 22 Own interpretation · 13 Not checked yet · 8 Own method · 5 No basis given · 1 Partly supported · accuracy not yet measured

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  • It may state two separate claims as one. 36
  • Probably not a substantial claim. 14
47 of 47

  • The paper relies on this without support.

    Mitochondrial quality control is essential in highly structured cells such as neurons and muscles.

    “Mitochondrial quality control is essential in highly structured cells such as neurons and muscles.”

  • The paper relies on this without support.

    Insulin signaling through mTOR plays a critical role in controlling protein synthesis.

    “Insulin signaling, through mTOR, plays a critical role in controlling protein synthesis.”

  • The paper relies on this without support.

    FGF21 causes GH resistance.

    “Because FGF21 causes GH resistance”

  • The authors state this as an assumption.

    The calibration equation and constants (KD = 145 nM, Rmin, Rmax, β) used to convert Fura-2 fluorescence ratios to free cytosolic calcium concentration are valid.

    “Under these assumptions, the resting cytosolic calcium concentration will be 71 nM at R = 0.87 and 87 nM at R = 0.92.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

2 of 2

Citations

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Metadata

Authors
Favaro, G., Romanello, V., Varanita, T., Andrea Desbats, M., Morbidoni, V., Tezze, C., Albiero, M., Canato, M., Gherardi, G., De Stefani, D., Mammucari, C., Blaauw, B., Boncompagni, S., Protasi, F., Reggiani, C., Scorrano, L., Salviati, L., & Sandri, M.
Affiliations
  • University of Padua (IT)
  • University of Chieti-Pescara (IT)
Corresponding author
  • Salviati, Leonardo
  • Sandri, Marco
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-06-12
Volume
10
Issue
1
Pages
2576-2576
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-10226-9
PMC
PMC6561930
OpenAlex
W2950169360
Semantic Scholar
f4a2921dcb6e0705b3925fc5a9a47319adb62545
Cited by
398
References
62
Citations by year
2026: 30 · 2025: 44 · 2024: 53 · 2023: 62 · 2022: 53 · 2021: 78 · 2020: 67 · 2019: 10
FWCI
17.20
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
Funders
  • Fondazione Cassa di Risparmio di Padova e Rovigo
  • European Commission (MSCA-RISE-2014, H2020, 645648, H2020‐MSCA‐RISE‐2014, H2020-MSCA, H2020 MSCA Rise)
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (133130)
  • Ministero della Salute (GR-2011-02352681)
  • Associazione Italiana per la Ricerca sul Cancro (PRIN 2015, 17388, PRIN 2010)
  • Horizon 2020 Framework Programme (H2020-MSCA-RISE-2014, H2020-MSCA, H2020-MSCA-RISE, 645648)
  • FP7 Ideas: European Research Council (282310)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
Keywords
Drp1, mitochondrial fission, mitochondrial morphology, calcium homeostasis, muscle atrophy, skeletal muscle, mitochondrial quality control, ubiquitin-proteasome system, unfolded protein response, myofiber degeneration, mitochondrial calcium uptake, sarcopenia, cancer cachexia
MeSH terms
Animals, Calcium, Cell Nucleus, Disease Models, Animal, Homeostasis, Humans, Mitochondria, Muscle, Ubiquitins, Mitochondrial Myopathies, Mice, Knockout, Muscle, Skeletal, Dynamins, Proteasome Endopeptidase Complex, Mice, Unfolded Protein Response, Proteolysis, Mitochondrial Dynamics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-10-06, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-10-06 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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